Photodynamic therapy (PDT) is an emerging modality for the treatment of neoplastic and non-neoplastic diseases. Using photodynamic therapeutic approaches, photosensitizers are localized in target tissues, and subsequently activated with an appropriate wavelength of light. Light activation of the photosensitizers generates active molecular species, such as free radicals and singlet oxygen (1O2), which are toxic to target cells and tissues. PDT is known to produce tumoricidal effects within malignant tissues. Tumors in virtually every anatomic site have been treated with PDT, and most are responsive to this therapy to some extent.
To date, several thousand patients have been treated with PDT for a variety of neoplasms. Randomized clinical trials of this modality were initiated in 1987, using a purified form of HPD, Photofrin® (Marcus, 1992), (Dougherty et al., 1998). These first randomized trials were sponsored by Quadra Logic Technologies, Inc. (now QLT Phototherapeutics, Vancouver, Canada) and American Cyanamid Co. (Pearl River, N.Y.), and compared the efficacy of PDT with that of other forms of therapy for bladder, esophageal, and lung cancers. Within the past 5 years, significant progress has been made worldwide in obtaining regulatory approval for a variety of indications. Currently, PDT with the photosensitizer Photofrin® is approved in at least 10 countries. Approval for treatment with other photosensitizers has been requested in the United States, Canada, and Europe.
PDT is a binary therapy, having the advantage of inherent dual selectivity. First, selectivity is achieved by an increased concentration of the photosensitizer in target tissue, and second, the irradiation can be limited to a specified volume. Provided that the photosensitizer is non-toxic, only the irradiated areas will be affected, even if the photosensitizer does bind to normal tissues. Selectivity thus obtained may be adequate for certain anatomical sites, such as skin and oral cavity, however, for more complex sites such as the peritoneal cavity, greater selectivity than that achievable with current photosensitizers is necessary, so that colateral damage to normal organs can be minimized. Selectivity can be even further enhanced by attaching photosensitizers to molecular delivery systems that have high affinity for target tissue (Hasan, 1992), (Strong et al., 1994). For example, one way to improve selectivity is to link the photosensitizer to a monoclonal antibody directed against cancer-associated antigens in an approach known as photoimmunotherapy (PIT). The resulting photoimmunoconjugate (PIC) delivers the photosensitizer directly to the tumor cell of interest.
The use of photosensitizer immunoconjugates (PICs) offers improved photosensitizer delivery specificity and could broaden the applicability of photodynamic therapy (PDT). For example, it has been suggested that PDT might be used effectively in the treatment of small diffuse malignancies present in a cavity, such as the peritoneum or bladder, if the photosensitizer could be made to accumulate with high specificity in malignant cells (Hamblin et al., 1996). This would allow photodynamic destruction of diseased cells while sparing adjacent normal tissues of sensitive organs.
Many monoclonal antibodies known in the art possess tumoricidal activity. The combined therapeutic use of a tumoricidal antibody and a photosensitizer compound is referred to herein as photodynamic combination therapy or “combination therapy.”Combination therapies advantageously co-localize photosensitizer compounds and tumoricidal antibodies in tumor tissue. Combination therapies would include PICs wherein the monoclonal antibody component has an inhibitory effect on tumor growth. The inhibitory effects of a combination therapy comprising a tumoricidal antibody and a photosensitizer compound on tumor growth were heretofore unknown.
Tumoricidal antibodies, when used as monotherapy for reducing tumor growth, can have associated toxicity. Combination therapies requiring reduced levels of antibody administration can also reduce the occurrence of associated toxicity.
For complicated diseases, such as those involving intraperitoneal cancers, combination therapies are likely to prevail over standard treatment regimes (Duska et al., 1999). Ovarian cancer is one example of an intraperitoneal cancer where combination therapies have the potential to be of great use. Ovarian cancer ranks as the fourth most common malignancy in American women, responsible for more deaths than any other cancer in the female reproductive tract (Greenlee et al., 2000; Ozols, 1994). Only 25% of cases are detected at a localized state, with most patients presenting with late stage disease. Reported 5-year survival for advanced ovarian cancer is 28% (Greenlee et al., 2000). This rather poor prognosis reflects the negligible effect that both advances in surgical technique and chemotherapy have had over the past ten years in the treatment of ovarian cancer. Currently, advanced disease is treated by staging/debulking surgery, followed by chemotherapy. Approximately 50% of patients will have documented positive second look laparotomies following first line treatment (Bolis et al., 1996). Among those women with negative second look laparotomies, 50% will present later with disease recurrence (Bolis et al., 1996). Recurrent disease is rarely curable, since there are currently no effective salvage treatments that affect survival. New treatments are necessary for the management of advanced and recurrent epithelial ovarian cancer; however, new therapeutic approaches have been difficult to develop. Use of PICs and/or combination therapies can offer a new therapeutic approach to the treatment of many cancers, including ovarian cancers.
Although nearly 20 years has past since PICs were first conceived (Mew et al., 1983), no clinically useful PICs yet exist. Improved PICs would not only be of use in combination therapies, but in any application of PDT wherein selective delivery and accumulation of photosensitizers to a target tissue is desired. This would include, for example, diagnostic methods using PICs. Literature reviews of PIC research (Hasan, 1992), (Sternberg et al., 1998), (Yarmush et al., 1993), (Savellano, 2000), have concluded that a major impasse encountered in this field has been the synthesis and purification of functional, well-characterized conjugates.
Several major problems with the design, synthesis, and purification of PICs have not been dealt with satisfactorily in PIC investigations. In particular, many previous studies of PICs did not thoroughly investigate whether the photodynamic effects of the conjugate preparations were due to the specific action of the conjugates or whether they were due to the action of noncovalently-associated free photosensitizer impurities present in the conjugate preparations (Hasan, 1992), (Sternberg et al., 1998), (Savellano, 2000). This issue is one of the most cumbersome problems encountered in PIC research. Due to the hydrophobic and/or highly adsorptive nature of most PDT-type photosensitizers, it has been very difficult to remove unconjugated photosensitizer impurities from PIC preparations. Moreover, for similar reasons, it has been difficult to maintain solubility of the PIC preparations. Whereas the best photosensitizers are usually hydrophobic and lipophilic, antibodies and immunoconjugates must remain water-soluble and disaggregated in order to reach their designated targets in an efficient manner via the circulation.
Thus, for the most part, problems in the art are attributable to the incompatible solubilities of photosensitizers and antibodies. Because previous studies of PICs have not utilized conjugation methods that are capable of circumventing the incompatible solubilities of photosensitizers and antibodies, the conjugate preparations very likely contained significant amounts of aggregated material and noncovalently-associated free photosensitizer impurities. Consequently, interpretation of the observed biological effects of the PICs in these studies has been difficult, especially in in vitro studies, since the effects of the actual conjugates cannot be clearly distinguished from the effects of noncovalently-associated free photosensitizer impurities and/or large aggregates that may have been present in the PIC preparations. In turn, it has been difficult to discern what measures must be taken to improve the overall performance of PIC constructs.
The use of benzoporphyrin derivative (BPD) photosensitizers in PICs is highly desirable. For example, BPD Verteporfin, has been thoroughly characterized, (Richter et al., 1987), (Aveline et al., 1994), and it has been found to be a highly potent photosensitizer for PDT. Investigations using BPD PICs have been extensively reported in the literature (Levy et al., 1989), (Jiang et al., 1990), (Jiang, 1993). However, similar to studies involving other photosensitizers, studies of BPD-based conjugates did not convincingly demonstrate that the photodynamic activity of the PIC preparations was predominantly due to the activity of the conjugates and not due to the activity of free BPD impurity present in the PIC preparations. In addition, the conjugation protocols described in the literature for linking BPD directly to antibodies failed to produce functional, high purity conjugate preparations.
Methods of producing improved PICs through the covalent linkage of photosensitizers to antibodies were heretofore unknown. PICs that are free of undesirable photosensitizer contaminants will further the development of improved photodynamic therapies, including combination therapies, and diagnostic methods.